Light scattering in biological tissue is a complex process that occurs as photons traverse index of refraction mismatches along their propagation path. The index mismatches are associated with tissue morphology (e.g. cytoskeletal arrangement) and cellular ultrastructure (e.g. size and shape of nucleus, mitochondria, other cytoplasmic organelles). Measurements of scattering remission spectra have shown sensitivity to sub-cellular morphological changes in biological tissue; these observations support the use of scattering as an endogenous and label-free contrast mechanism to differentiate between tissue types. Scattering spectroscopy has important clinical implications for the diagnosis of cancers, and for the assessment of surgical margins to guide tumor resections.
While scatter remission spectra are sensitive to biological structure and morphology, the biological information that is encoded in collected spectra is dependent on the light transport regime that is sampled. Scattering interactions between photons and tissue can be described by a basic set of parameters including (a) the frequency of scattering events characterized by the scattering coefficient μs, (b) the probability of scattering angles θs defined by the scattering phase function P(θs), and (c) the average scatter direction characterized by the first moment g1=<cos θs> of the scattering phase function P(θs). Photons that have experienced many scattering events within turbid media have lost the orientation to their original direction of travel and are considered diffuse. Diffuse remission is insensitive to the direction of individual scattering events, and can be modeled with a diffusion approximation to the radiation transport equation, which introduces the reduced scattering coefficient μ′s=μs(1−g1) to combine the effects of scatter frequency and directionality into a single lumped parameter. Diffusion theory is generally applicable to light that remits one or two reduced scattering lengths, i.e., 1-2 (μs)−1, from the source location, which in biological tissue is approximately 1-2 millimeters.
Studies have correlated diffuse measurements of μ′s(λ) with the size distribution of scattering centers in bulk tissue, providing a noninvasive characterization of biological tissue structure. However, such prior measurements are averaged over a large tissue volume and are insensitive to changes in local tissue microstructure.
Localized measurements of scatter remission have been developed to interrogate small tissue volumes of interest. When near the source, these prior art measurements collect a population of photons that have experienced few scattering events, making the signal sensitive to the direction of individual scattering events. Light in this transport regime is termed sub-diffuse. Model-based interpretation of sub-diffuse remission spectra requires both μ′s and a parameter that describes the phase function-dependent probability of large-angle backscatter events that are likely to be collected during reflectance measurements. For forward-directed scattering media, such as in biological tissue, the relative probability of large backscattering events is proportional to the weighted ratio of the 1st and 2nd Legendre moments, g1 and g2, respectively, of P(θs). This probability is given by γ=(1−g2)/(1−g1). So far, approaches that have quantitated sub-diffusion scattering parameters in biological tissue have classically been limited to the sampling of small volumes, usually sub-millimeters. In the prior art, imaging of localized scatter has been achieved by mechanically scanning a fiber optic, and results suggest that contextual interpretation of heterogeneous spatial-variations in scatter remission may discriminate between tissue types and potentially guide clinical decisions. However, these prior-art approaches can be time intensive, and studies published to date have not interpreted the signal in terms of underlying scattering properties.
Guidance of clinical decisions, for example during surgery, often requires fast assessment of large areas of tissue. This requirement has limited the translation and adoption of localized quantitative spectroscopic approaches within the clinical theatre. Recently, spatial frequency domain imaging (SFDI) has been demonstrated as a method to provide quantitative spatial maps of μ′s and the absorption coefficient μa, in turbid media, with fast image acquisitions over a wide field of view. See Cuccia et al. in “Modulated imaging: quantitative analysis and tomography of turbid media in the spatial-frequency domain”, Opt. Lett. 30(11), 1354-1356 (2005), Cuccia et al. in “Quantitation and mapping of tissue optical properties using modulated imaging”, J. Biomed. Op. 14(2), 024012 (2009), and Gioux et al. in “First-in-human pilot study of spatial frequency domain oxygenation imaging system”, J. Biomemd. Opt. 16(8), 086015 (2011). This method applies structured light to illuminate the surface of a medium with sinusoidal intensity patterns at various spatial frequencies fx. The collected signal is demodulated and optical properties are estimated from diffusion theory. The diffuse analysis invokes two important assumptions: (a) scatter dominates absorption such that μ′s>>μa, and (b) the maximum collected spatial frequency fx is limited to 0.25μtr to 0.33μtr (where μtr=μ′s for the non-absorbing case), a range of spatial frequencies that limits the sampling of photons which experience few scattering events. The first assumption has been addressed by a Monte Carlo look up table to analyze SFDI signals in highly absorbing tissues. To date, no study has directly addressed the second assumption and quantitatively analyzed sub-diffuse remission collected from structured illumination imaging, although Konecky et al., in “Imaging scattering orientation with spatial frequency domain imaging”, J. Biomed. Opt. 16(12), 126001 (2011), considered rotation of the incident illumination pattern to identify directional preferences for scatter within a wide field of view. This unique illumination pattern was characterized as a special case of diffuse light collection that was sensitive to the anisotropic orientation of scatterers on the order of the transport length in the sampled medium, but did not yield estimates of quantitative scattering parameters.